人—牛异种体细胞克隆胚胎构建及其线粒体来源检测
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摘要
人-动物异种间体细胞核移植技术可利用动物的卵母细胞为核受体,以人类的体细胞为核供体构建异种间体细胞克隆胚,通过体外培养获取人源性胚胎干细胞。这项技术为获得人类干细胞以及人类器官移植开辟了一条崭新的途径。但极低的成功率限制了这项技术在实践中的应用,这可能归因于体细胞核在异种卵母细胞中未能完全再程序化。同时,异种间克隆胚存在广泛的线粒体杂合现象,即来源于供受体细胞的两套线粒体共存。影响异种间克隆胚及克隆后代中线粒体杂合性的因素还不清楚。动物源性线粒体的存在,是人-动物异种体细胞克隆胚胎应用于实践的一大障碍。
本实验以体外培养成熟的牛卵母细胞为核受体,分别以人胎儿成纤维细胞(HFFs)、牛胎儿成纤维细胞(BFFs)为核供体构建同异种间体细胞克隆胚,通过体外培养观察两者在发育能力方面的差异。此外,在早期发育的不同阶段(2-、4-、8-、桑椹胚、囊胚),采用PCR的方法检测了异种间克隆胚中供受体来源的线粒体DNA(mtDNA)存在情况,以及异种克隆胚胎核基因来源情况。
人-牛异种克隆胚胎构建试验中发现,试验的批次效应明显,说明卵母细胞与供体细胞的协调性有很强的随机性,两者的协调性也是影响克隆效率的最关键因素。
同异种间克隆胚的体外发育结果表明,同种间克隆胚的体外囊胚发育率(10.1%)高于异种间克隆胚的体外囊胚发育率(2.88%),且这种差异主要是由异种间克隆胚在8-细胞到桑椹胚期间的发育能力显著低于同种间克隆胚(p＜0.05)造成的。
对异种间克隆胚核基因来源的检测表明,异种间克隆胚的核基因来源于人类供体细胞。
对不同发育阶段异种间克隆胚中供受体来源的mtDNA检测结果表明人线粒体DNA存在于桑椹胚之前的各个胚胎发育阶段,牛线粒体DNA存在于整个植入前的胚胎发育阶段。
The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animals oocytes as nuclear recipient and human somatic cell as nuclear donor, can afford more opportunities in human embryonic stem cell and human organ transplantation by obtaining embryonic stem cell from interspecies cloned embryos which were cultured in vitro. But the application of this technique was limited by its extremely low efficiency which maybe can be attributed to donor nucleus not fully reprogrammed by xenogenic recipient cytoplasmy. Moreover, two types of mitochondria, which were derived respectively from nuclear donor and nuclear recipient, were observed in many interspecies cloned embryos and offspring. But the factors which affected mitochondrial heteroplasmy were still unclear. It should be considered that allogeneic mitochondria present in NT-ESC or NT-ESC derived cells could be recognized by the host immune system, leading to disrupted mitochondrial membrane potential that induces the apoptotic cell signaling pathway, thus leading to cell death.
In present study, intra- and inerspecies cloned embryos were constructed using in vitro matured bovine oocytes as nuclear recipient and human- (HFFs) or bovine fetal fibroblasts (BFFs) as nuclear donor. The difference in developmental ability between intra- and interspecies cloned embryos were investigated by cultured them in vitro. Moreover, at various developmental stage (2-, 4-, 8-, morula and blastocyst) of interspecies cloned embryos, the percentage of two types of mtDNA and mtRNA was examined using PCR.
A significant batch effect was found in construction of interspecies embryos. This showed that the cooperation between the enucleated oocytes and the donor cell is random. The cooperation is the most important factor in somatic cell nuclear transfer.
The result of development in vitro showed that the rate of intra- blastocyst (10.1%) was higher than that of interspecies blastocyst (2.88%), and this difference was mainly due to lower developmental ability (p<0.05) of intra- than that of interspecies cloned embryos during 8- to morula.
The results of analyzing on karyogene of interspecies embryos showed that the karyogene of interspecies embryos is from human donor cell.
The results of examining two types of mitochondria in interspecies cloned embryos showed that before morula stage, the mtDNA from HFF can existed in interspecies cloned embryos; at all developmental stage, the mtDNA from bovine oocyte can existed in interspecies cloned embryos.
本实验以体外培养成熟的牛卵母细胞为核受体,分别以人胎儿成纤维细胞(HFFs)、牛胎儿成纤维细胞(BFFs)为核供体构建同异种间体细胞克隆胚,通过体外培养观察两者在发育能力方面的差异。此外,在早期发育的不同阶段(2-、4-、8-、桑椹胚、囊胚),采用PCR的方法检测了异种间克隆胚中供受体来源的线粒体DNA(mtDNA)存在情况,以及异种克隆胚胎核基因来源情况。
人-牛异种克隆胚胎构建试验中发现,试验的批次效应明显,说明卵母细胞与供体细胞的协调性有很强的随机性,两者的协调性也是影响克隆效率的最关键因素。
同异种间克隆胚的体外发育结果表明,同种间克隆胚的体外囊胚发育率(10.1%)高于异种间克隆胚的体外囊胚发育率(2.88%),且这种差异主要是由异种间克隆胚在8-细胞到桑椹胚期间的发育能力显著低于同种间克隆胚(p＜0.05)造成的。
对异种间克隆胚核基因来源的检测表明,异种间克隆胚的核基因来源于人类供体细胞。
对不同发育阶段异种间克隆胚中供受体来源的mtDNA检测结果表明人线粒体DNA存在于桑椹胚之前的各个胚胎发育阶段,牛线粒体DNA存在于整个植入前的胚胎发育阶段。
The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animals oocytes as nuclear recipient and human somatic cell as nuclear donor, can afford more opportunities in human embryonic stem cell and human organ transplantation by obtaining embryonic stem cell from interspecies cloned embryos which were cultured in vitro. But the application of this technique was limited by its extremely low efficiency which maybe can be attributed to donor nucleus not fully reprogrammed by xenogenic recipient cytoplasmy. Moreover, two types of mitochondria, which were derived respectively from nuclear donor and nuclear recipient, were observed in many interspecies cloned embryos and offspring. But the factors which affected mitochondrial heteroplasmy were still unclear. It should be considered that allogeneic mitochondria present in NT-ESC or NT-ESC derived cells could be recognized by the host immune system, leading to disrupted mitochondrial membrane potential that induces the apoptotic cell signaling pathway, thus leading to cell death.
In present study, intra- and inerspecies cloned embryos were constructed using in vitro matured bovine oocytes as nuclear recipient and human- (HFFs) or bovine fetal fibroblasts (BFFs) as nuclear donor. The difference in developmental ability between intra- and interspecies cloned embryos were investigated by cultured them in vitro. Moreover, at various developmental stage (2-, 4-, 8-, morula and blastocyst) of interspecies cloned embryos, the percentage of two types of mtDNA and mtRNA was examined using PCR.
A significant batch effect was found in construction of interspecies embryos. This showed that the cooperation between the enucleated oocytes and the donor cell is random. The cooperation is the most important factor in somatic cell nuclear transfer.
The result of development in vitro showed that the rate of intra- blastocyst (10.1%) was higher than that of interspecies blastocyst (2.88%), and this difference was mainly due to lower developmental ability (p<0.05) of intra- than that of interspecies cloned embryos during 8- to morula.
The results of analyzing on karyogene of interspecies embryos showed that the karyogene of interspecies embryos is from human donor cell.
The results of examining two types of mitochondria in interspecies cloned embryos showed that before morula stage, the mtDNA from HFF can existed in interspecies cloned embryos; at all developmental stage, the mtDNA from bovine oocyte can existed in interspecies cloned embryos.
引文
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